The CRISPR/Cas9 system has enabled tremendous advances in genome editing in many organisms, versatile gene expression control, and imaging of genomic loci in living cells. This R21 bioengineering project aims to advance the applications of CRISPR/Cas9 by generating new Cas9 proteins that are orthogonal to each other and can therefore be used together but for different, controllable purposes. This would make many new applications possible that require efficient multiplexing of distinct functions, such as simultaneous control of many genes that need to be either activated or repressed, or multicolor imaging of different DNA loci in live cells. The current system consists of the Cas9 protein and a small guide RNA (sgRNA), which can be programmed to recognize any arbitrary DNA sequence that is complimentary to the sgRNA and contains a protospacer adjacent motif (PAM) recognized by Cas9. To date, the Cas9 protein from S. pyogenes (SpCas9) has been used almost ubiquitously, due to its robustness and flexibility. The proposed new Cas9 tools are based on SpCas9 but have additional specificity elements (new PAM sequences) that can direct distinct functionalities (defined by different PAMs) to the target sequences (programmed as before by the sgRNA). Previous protein engineering has failed to generate new Cas9 tools with substantially altered PAM specificity, which likely requires the structure of the SpCas9 active site to be remodeled considerably. To overcome this bottleneck, we will utilize computational strategies borrowed from the field of robotics, which we have previously shown can model protein conformations with atomic accuracy. We will integrate these methods for the first time with design, to alter active site structures for new functions. In Aim 1, we will develop new computational design methods to accurately position key sidechains by remodeling the backbone of the surrounding functional site environment, test these methods on recapitulating functional site conformations in existing structures, and make forward predictions to design SpCas9 variants that recognize new PAMs. In Aim 2, we will test and optimize these SpCas9 variants and apply them to multicolor imaging of defined DNA loci in human cells. To achieve these goals, our proposal brings together two investigators who are experts in computational protein design (Kortemme) and fluorescence microscopy (Huang). Both the proposed design method and the design application are innovative, but supported by preliminary data showing labeling of genomic loci by an SpCas9 variant computationally designed to recognize an altered PAM. To further address risks, we plan to complement computation with experimental testing of sequence libraries and optimization of design candidates by directed evolution. The outcomes of this project would advance both (i) computational design to address more complex engineering goals than are possible now, and (ii) CRISPR/Cas9 applications by allowing simultaneous control of multiple functions, including systematic analysis of gene function in health and disease, and dynamic imaging of fundamental processes in living cells.